The association between hemodynamics and wall characteristics in human intracranial aneurysms: a review.

Hemodynamics plays a key role in the natural history of intracranial aneurysms (IAs). However, studies exploring the association between aneurysmal hemodynamics and the biological and mechanical characteristics of the IA wall in humans are sparse. In this review, we survey the current body of literature, summarize the studies' methodologies and findings, and assess the degree of consensus among them. We used PubMed to perform a systematic review of studies that explored the association between hemodynamics and human IA wall features using different sources. We identified 28 publications characterizing aneurysmal flow and the IA wall: 4 using resected tissues, 17 using intraoperative images, and 7 using vessel wall magnetic resonance imaging (MRI). Based on correlation to IA tissue, higher flow conditions, such as high wall shear stress (WSS) with complex pattern and elevated pressure, were associated with degenerated walls and collagens with unphysiological orientation and faster synthesis. MRI studies strongly supported that low flow, characterized by low WSS and high blood residence time, was associated with thicker walls and post-contrast enhancement. While significant discrepancies were found among those utilized intraoperative images, they generally supported that thicker walls coexist at regions with prolonged residence time and that thinner regions are mainly exposed to higher pressure with complex WSS patterns. The current body of literature supports a theory of two general hemodynamic-biologic mechanisms for IA development. One, where low flow conditions are associated with thickening and atherosclerotic-like remodeling, and the other where high and impinging flow conditions are related to wall degeneration, thinning, and collagen remodeling.

Endogenous animal models of intracranial aneurysm development: a review.

The pathogenesis and natural history of intracranial aneurysm (IA) remains poorly understood. To this end, animal models with induced cerebral vessel lesions mimicking human aneurysms have provided the ability to greatly expand our understanding. In this review, we comprehensively searched the published literature to identify studies that endogenously induced IA formation in animals. Studies that constructed aneurysms (i.e., by surgically creating a sac) were excluded. From the eligible studies, we reported information including the animal species, method for aneurysm induction, aneurysm definitions, evaluation methods, aneurysm characteristics, formation rate, rupture rate, and time course. Between 1960 and 2019, 174 articles reported endogenous animal models of IA. The majority used flow modification, hypertension, and vessel wall weakening (i.e., elastase treatment) to induce IAs, primarily in rats and mice. Most studies utilized subjective or qualitative descriptions to define experimental aneurysms and histology to study them. In general, experimental IAs resembled the pathobiology of the human disease in terms of internal elastic lamina loss, medial layer degradation, and inflammatory cell infiltration. After the early 2000s, many endogenous animal models of IA began to incorporate state-of-the-art technology, such as gene expression profiling and 9.4-T magnetic resonance imaging (MRI) in vivo imaging, to quantitatively analyze the biological mechanisms of IA. Future studies aimed at longitudinally assessing IA pathobiology in models that incorporate aneurysm growth will likely have the largest impact on our understanding of the disease. We believe this will be aided by high-resolution, small animal, survival imaging, in situ live-cell imaging, and next-generation omics technology.

Classification models using circulating neutrophil transcripts can detect unruptured intracranial aneurysm.

BACKGROUND: Intracranial aneurysms (IAs) are dangerous because of their potential to rupture. We previously found significant RNA expression differences in circulating neutrophils between patients with and without unruptured IAs and trained machine learning models to predict presence of IA using 40 neutrophil transcriptomes. Here, we aim to develop a predictive model for unruptured IA using neutrophil transcriptomes from a larger population and more robust machine learning methods. METHODS: Neutrophil RNA extracted from the blood of 134 patients (55 with IA, 79 IA-free controls) was subjected to next-generation RNA sequencing. In a randomly-selected training cohort (n = 94), the Least Absolute Shrinkage and Selection Operator (LASSO) selected transcripts, from which we constructed prediction models via 4 well-established supervised machine-learning algorithms (K-Nearest Neighbors, Random Forest, and Support Vector Machines with Gaussian and cubic kernels). We tested the models in the remaining samples (n = 40) and assessed model performance by receiver-operating-characteristic (ROC) curves. Real-time quantitative polymerase chain reaction (RT-qPCR) of 9 IA-associated genes was used to verify gene expression in a subset of 49 neutrophil RNA samples. We also examined the potential influence of demographics and comorbidities on model prediction. RESULTS: Feature selection using LASSO in the training cohort identified 37 IA-associated transcripts. Models trained using these transcripts had a maximum accuracy of 90% in the testing cohort. The testing performance across all methods had an average area under ROC curve (AUC) = 0.97, an improvement over our previous models. The Random Forest model performed best across both training and testing cohorts. RT-qPCR confirmed expression differences in 7 of 9 genes tested. Gene ontology and IPA network analyses performed on the 37 model genes reflected dysregulated inflammation, cell signaling, and apoptosis processes. In our data, demographics and comorbidities did not affect model performance. CONCLUSIONS: We improved upon our previous IA prediction models based on circulating neutrophil transcriptomes by increasing sample size and by implementing LASSO and more robust machine learning methods. Future studies are needed to validate these models in larger cohorts and further investigate effect of covariates.

Biomarkers from circulating neutrophil transcriptomes have potential to detect unruptured intracranial aneurysms.

BACKGROUND: Intracranial aneurysms (IAs) are dangerous because of their potential to rupture and cause deadly subarachnoid hemorrhages. Previously, we found significant RNA expression differences in circulating neutrophils between patients with unruptured IAs and aneurysm-free controls. Searching for circulating biomarkers for unruptured IAs, we tested the feasibility of developing classification algorithms that use neutrophil RNA expression levels from blood samples to predict the presence of an IA. METHODS: Neutrophil RNA extracted from blood samples from 40 patients (20 with angiography-confirmed unruptured IA, 20 angiography-confirmed IA-free controls) was subjected to next-generation RNA sequencing to obtain neutrophil transcriptomes. In a randomly-selected training cohort of 30 of the 40 samples (15 with IA, 15 controls), we performed differential expression analysis. Significantly differentially expressed transcripts (false discovery rate < 0.05, fold change ≥ 1.5) were used to construct prediction models for IA using four well-known supervised machine-learning approaches (diagonal linear discriminant analysis, cosine nearest neighbors, nearest shrunken centroids, and support vector machines). These models were tested in a testing cohort of the remaining 10 neutrophil samples from the 40 patients (5 with IA, 5 controls), and model performance was assessed by receiver-operating-characteristic (ROC) curves. Real-time quantitative polymerase chain reaction (PCR) was used to corroborate expression differences of a subset of model transcripts in neutrophil samples from a new, separate validation cohort of 10 patients (5 with IA, 5 controls). RESULTS: The training cohort yielded 26 highly significantly differentially expressed neutrophil transcripts. Models using these transcripts identified IA patients in the testing cohort with accuracy ranging from 0.60 to 0.90. The best performing model was the diagonal linear discriminant analysis classifier (area under the ROC curve = 0.80 and accuracy = 0.90). Six of seven differentially expressed genes we tested were confirmed by quantitative PCR using isolated neutrophils from the separate validation cohort. CONCLUSIONS: Our findings demonstrate the potential of machine-learning methods to classify IA cases and create predictive models for unruptured IAs using circulating neutrophil transcriptome data. Future studies are needed to replicate these findings in larger cohorts.

Differential gene expression by endothelial cells under positive and negative streamwise gradients of high wall shear stress.

Flow impingement at arterial bifurcations causes high frictional force [or wall shear stress (WSS)], and flow acceleration and deceleration in the branches create positive and negative streamwise gradients in WSS (WSSG), respectively. Intracranial aneurysms tend to form in regions with high WSS and positive WSSG. However, little is known about the responses of endothelial cells (ECs) to either positive or negative WSSG under high WSS conditions. We used cDNA microarrays to profile gene expression in cultured ECs exposed to positive or negative WSSG for 24 h in a flow chamber where WSS varied between 3.5 and 28.4 Pa. Gene ontology and biological pathway analysis indicated that positive WSSG favored proliferation, apoptosis, and extracellular matrix processing while decreasing expression of proinflammatory genes. To determine if similar responses occur in vivo, we examined EC proliferation and expression of the matrix metalloproteinase ADAMTS1 under high WSS and WSSG created at the basilar terminus of rabbits after bilateral carotid ligation. Precise hemodynamic conditions were determined by computational fluid dynamic simulations from three-dimensional angiography and mapped on immunofluorescence staining for the proliferation marker Ki-67 and ADAMTS1. Both proliferation and ADAMTS1 were significantly higher in ECs under positive WSSG than in adjacent regions of negative WSSG. Our results indicate that WSSG elicits distinct EC gene expression profiles and particular biological pathways including increased cell proliferation and matrix processing. Such EC responses may be important in understanding the mechanisms of intracranial aneurysm initiation at regions of high WSS and positive WSSG.

A role for microtubules in endothelial cell protrusion in three-dimensional matrices.

BACKGROUND INFORMATION: Most cells reside in vivo in a three-dimensional (3D) environment surrounded by extracellular matrix and other neighbouring cells, conditions that are different from those found by cells cultured in vitro on two-dimensional (2D) substrata. Cell morphology and behaviour are very different under these two different conditions, but the structural basis for these differences is still not understood, especially the role of microtubules (MTs). To address this issue, we studied the early spreading behaviour of bovine aortic endothelial cells (BAECs) cultured in 3D collagen matrices and on 2D substrata, in the presence of MT-disrupting drugs. RESULTS: We found that depolymerisation of MTs greatly reduces the ability of BAECs to form large and stable protrusions inside 3D collagen matrices, an effect that is less pronounced when the cells are cultured on 2D substrata. Colcemid-treated BAECs inside 3D matrices begin assembling protrusions and pull on the matrix, but they fail to extend those protrusions deep into the matrix. It has been previously reported that MT disruption affects Rho signalling which may result in increased cell rigidity and adhesiveness to 2D matrices. Accordingly, we demonstrate that colcemid treatment indeed leads to activation of Rho-kinase (ROCK) targets, which in turn results in activation of regulatory myosin light chains, and that blocking of ROCK mitigates some of the effects of MT disruption in cell spreading in 3D. CONCLUSIONS: Our results show that MT depolymerisation is particularly disruptive when cells interact with pliable 3D matrices, suggesting a role for MTs and the Rho pathway in the fine-tuning of contractile and adhesive forces necessary to sustain cell motility in vivo.

Survivin as a mediator of stiffness-induced cell cycle progression and proliferation of vascular smooth muscle cells.

Stiffened arteries are a pathology of atherosclerosis, hypertension, and coronary artery disease and a key risk factor for cardiovascular disease events. The increased stiffness of arteries triggers a phenotypic switch, hypermigration, and hyperproliferation of vascular smooth muscle cells (VSMCs), leading to neointimal hyperplasia and accelerated neointima formation. However, the mechanism underlying this trigger remains unknown. Our analyses of whole-transcriptome microarray data from mouse VSMCs cultured on stiff hydrogels simulating arterial pathology identified 623 genes that were significantly and differentially expressed (360 upregulated and 263 downregulated) relative to expression in VSMCs cultured on soft hydrogels. Functional enrichment and gene network analyses revealed that these stiffness-sensitive genes are linked to cell cycle progression and proliferation. Importantly, we found that survivin, an inhibitor of apoptosis protein, mediates stiffness-dependent cell cycle progression and proliferation as determined by gene network and pathway analyses, RT-qPCR, immunoblotting, and cell proliferation assays. Furthermore, we found that inhibition of cell cycle progression did not reduce survivin expression, suggesting that survivin functions as an upstream regulator of cell cycle progression and proliferation in response to ECM stiffness. Mechanistically, we found that the stiffness signal is mechanotransduced via the FAK-E2F1 signaling axis to regulate survivin expression, establishing a regulatory pathway for how the stiffness of the cellular microenvironment affects VSMC behaviors. Overall, our findings indicate that survivin is necessary for VSMC cycling and proliferation and plays a role in regulating stiffness-responsive phenotypes.

Survivin regulates intracellular stiffness and extracellular matrix production in vascular smooth muscle cells.

Vascular dysfunction is a common cause of cardiovascular diseases characterized by the narrowing and stiffening of arteries, such as atherosclerosis, restenosis, and hypertension. Arterial narrowing results from the aberrant proliferation of vascular smooth muscle cells (VSMCs) and their increased synthesis and deposition of extracellular matrix (ECM) proteins. These, in turn, are modulated by arterial stiffness, but the mechanism for this is not fully understood. We found that survivin is an important regulator of stiffness-mediated ECM synthesis and intracellular stiffness in VSMCs. Whole-transcriptome analysis and cell culture experiments showed that survivin expression is upregulated in injured femoral arteries in mice and in human VSMCs cultured on stiff fibronectin-coated hydrogels. Suppressed expression of survivin in human VSMCs significantly decreased the stiffness-mediated expression of ECM components related to arterial stiffening, such as collagen-I, fibronectin, and lysyl oxidase. By contrast, expression of these ECM proteins was rescued by ectopic expression of survivin in human VSMCs cultured on soft hydrogels. Interestingly, atomic force microscopy analysis showed that suppressed or ectopic expression of survivin decreases or increases intracellular stiffness, respectively. Furthermore, we observed that inhibiting Rac and Rho reduces survivin expression, elucidating a mechanical pathway connecting intracellular tension, mediated by Rac and Rho, to survivin induction. Finally, we found that survivin inhibition decreases FAK phosphorylation, indicating that survivin-dependent intracellular tension feeds back to maintain signaling through FAK. These findings suggest a novel mechanism by which survivin potentially modulates arterial stiffness.

Endothelial cells express a unique transcriptional profile under very high wall shear stress known to induce expansive arterial remodeling.

Chronic high flow can induce arterial remodeling, and this effect is mediated by endothelial cells (ECs) responding to wall shear stress (WSS). To assess how WSS above physiological normal levels affects ECs, we used DNA microarrays to profile EC gene expression under various flow conditions. Cultured bovine aortic ECs were exposed to no-flow (0 Pa), normal WSS (2 Pa), and very high WSS (10 Pa) for 24 h. Very high WSS induced a distinct expression profile compared with both no-flow and normal WSS. Gene ontology and biological pathway analysis revealed that high WSS modulated gene expression in ways that promote an anti-coagulant, anti-inflammatory, proliferative, and promatrix remodeling phenotype. A subset of characteristic genes was validated using quantitative polymerase chain reaction: very high WSS upregulated ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motif-1), PLAU (urokinase plasminogen activator), PLAT (tissue plasminogen activator), and TIMP3, all of which are involved in extracellular matrix processing, with PLAT and PLAU also contributing to fibrinolysis. Downregulated genes included CXCL5 and IL-8 and the adhesive glycoprotein THBS1 (thrombospondin-1). Expressions of ADAMTS1 and uPA proteins were assessed by immunhistochemistry in rabbit basilar arteries experiencing increased flow after bilateral carotid artery ligation. Both proteins were significantly increased when WSS was elevated compared with sham control animals. Our results indicate that very high WSS elicits a unique transcriptional profile in ECs that favors particular cell functions and pathways that are important in vessel homeostasis under increased flow. In addition, we identify specific molecular targets that are likely to contribute to adaptive remodeling under elevated flow conditions.

Cellular and molecular responses of the basilar terminus to hemodynamics during intracranial aneurysm initiation in a rabbit model.

BACKGROUND/AIMS: Hemodynamics constitute a critical factor in the formation of intracranial aneurysms. However, little is known about how intracranial arteries respond to hemodynamic insult and how that response contributes to aneurysm formation. We examined early cellular responses at rabbit basilar termini exposed to hemodynamic insult that initiates aneurysmal remodeling. METHODS: Flow in the basilar artery was increased by bilateral carotid artery ligation. After 2 and 5 days, basilar terminus tissue was examined by immunohistochemistry and quantitative PCR. RESULTS: Within 2 days of flow increase, internal elastic lamina (IEL) was lost in the periapical region of the bifurcation, which experienced high wall shear stress and positive wall shear stress gradient. Overlying endothelium was still largely present in this region. IEL loss was associated with localized apoptosis and elevated expression of matrix metalloproteinases (MMPs) 2 and 9. A small number of inflammatory cells were sporadically scattered in the bifurcation adventitia and were not concentrated in regions of IEL loss and MMP elevation. Elevated MMP expression colocalized with smooth muscle α-actin in the media. CONCLUSION: The initial vascular response to aneurysm-initiating hemodynamic insult includes localized matrix degradation and cell apoptosis. Such destructive remodeling arises from intrinsic mural cells, rather than through inflammatory cell infiltration.

RNA Sequencing Data from Human Intracranial Aneurysm Tissue Reveals a Complex Inflammatory Environment Associated with Rupture.

BACKGROUND: Intracranial aneurysm (IA) rupture leads to deadly subarachnoid hemorrhages. However, the mechanisms leading to rupture remain poorly understood. Altered gene expression within IA tissue is linked to the pathobiology of aneurysm development and progression. Here, we analyzed expression patterns of control tissue samples and compared them to those of unruptured and ruptured IA tissue samples using data from the Gene Expression Omnibus (GEO). METHODS: FASTQ files for 21 ruptured IAs, 21 unruptured IAs, and 16 control tissue samples were accessed from the GEO database. DESeq2 was used for differential expression analysis in three comparisons: unruptured IA versus control, ruptured IA versus control, and ruptured versus unruptured IA. Genes that were differentially expressed in multiple comparisons were evaluated to find those progressively increasing/decreasing from control to unruptured to ruptured. Significance was tested by either analysis of variance/Gabriel or Brown-Forsythe/Games Howell (p < 0.05 was considered significant). We used additional RNA sequencing and proteomics datasets to evaluate if our differentially expressed genes (DEGs) were present in other studies. Bioinformatics analyses were performed with g:Profiler and Ingenuity Pathway Analysis. RESULTS: In total, we identified 1768 DEGs, of which 318 were found in multiple comparisons. Unruptured versus control reflected vascular remodeling processes, while ruptured versus control reflected inflammatory responses and cell activation/signaling. When comparing ruptured to unruptured IAs, we found massive activation of inflammation, inflammatory responses, and leukocyte responses. Of the 318 genes in multiple comparisons, 127 were found to be significant in the multi-cohort correlation analysis. Those that progressively increased (70 genes) were associated with immune system processes, while those that progressively decreased (38 genes) did not return any gene ontology terms. Many of our DEGs were also found in the other IA tissue sequencing studies. CONCLUSIONS: We found unruptured IAs relate more to remodeling processes, while ruptured IAs reflect more inflammatory and immune responses.

Identification of intima-to-media signals for flow-induced vascular remodeling using correlative gene expression analysis.

Changes in blood flow can induce arterial remodeling. Intimal cells sense flow and send signals to the media to initiate remodeling. However, the nature of such intima-media signaling is not fully understood. To identify potential signals, New Zealand white rabbits underwent bilateral carotid ligation to increase flow in the basilar artery or sham surgery (n = 2 ligated, n = 2 sham). Flow was measured by transcranial Doppler ultrasonography, vessel geometry was determined by 3D angiography, and hemodynamics were quantified by computational fluid dynamics. 24 h post-surgery, the basilar artery and terminus were embedded for sectioning. Intima and media were separately microdissected from the sections, and whole transcriptomes were obtained by RNA-seq. Correlation analysis of expression across all possible intima-media gene pairs revealed potential remodeling signals. Carotid ligation increased flow in the basilar artery and terminus and caused differential expression of 194 intimal genes and 529 medial genes. 29,777 intima-media gene pairs exhibited correlated expression. 18 intimal genes had > 200 medial correlates and coded for extracellular products. Gene ontology of the medial correlates showed enrichment of organonitrogen metabolism, leukocyte activation/immune response, and secretion/exocytosis processes. This demonstrates correlative expression analysis of intimal and medial genes can reveal novel signals that may regulate flow-induced arterial remodeling.

A machine learning pipeline revealing heterogeneous responses to drug perturbations on vascular smooth muscle cell spheroid morphology and formation.

Machine learning approaches have shown great promise in biology and medicine discovering hidden information to further understand complex biological and pathological processes. In this study, we developed a deep learning-based machine learning algorithm to meaningfully process image data and facilitate studies in vascular biology and pathology. Vascular injury and atherosclerosis are characterized by neointima formation caused by the aberrant accumulation and proliferation of vascular smooth muscle cells (VSMCs) within the vessel wall. Understanding how to control VSMC behaviors would promote the development of therapeutic targets to treat vascular diseases. However, the response to drug treatments among VSMCs with the same diseased vascular condition is often heterogeneous. Here, to identify the heterogeneous responses of drug treatments, we created an in vitro experimental model system using VSMC spheroids and developed a machine learning-based computational method called HETEROID (heterogeneous spheroid). First, we established a VSMC spheroid model that mimics neointima-like formation and the structure of arteries. Then, to identify the morphological subpopulations of drug-treated VSMC spheroids, we used a machine learning framework that combines deep learning-based spheroid segmentation and morphological clustering analysis. Our machine learning approach successfully showed that FAK, Rac, Rho, and Cdc42 inhibitors differentially affect spheroid morphology, suggesting that multiple drug responses of VSMC spheroid formation exist. Overall, our HETEROID pipeline enables detailed quantitative drug characterization of morphological changes in neointima formation, that occurs in vivo, by single-spheroid analysis.

Characterization of critical hemodynamics contributing to aneurysmal remodeling at the basilar terminus in a rabbit model.

BACKGROUND AND PURPOSE: Hemodynamic insult by bilateral common carotid artery ligation has been shown to induce aneurysmal remodeling at the basilar terminus in a rabbit model. To characterize critical hemodynamics that initiate this remodeling, we applied a novel hemodynamics-histology comapping technique. METHODS: Eight rabbits received bilateral common carotid artery ligation to increase basilar artery flow. Three underwent sham operations. Hemodynamic insult at the basilar terminus was assessed by computational fluid dynamics. Bifurcation tissue was harvested on day 5; histology was comapped with initial postligation hemodynamic fields of wall shear stress (WSS) and WSS gradient. RESULTS: All bifurcations showed internal elastic lamina loss in periapical regions exposed to accelerating flow with high WSS and positive WSS gradient. Internal elastic lamina damage happened 100% of the time at locations where WSS was >122 Pa and WSS gradient was >530 Pa/mm. The degree of destructive remodeling accounting for internal elastic lamina loss, medial thinning, and luminal bulging correlated with the magnitude of the hemodynamic insult. CONCLUSIONS: Aneurysmal remodeling initiates when local hemodynamic forces exceed specific limits at the rabbit basilar terminus. A combination of high WSS and positive WSS gradient represents dangerous hemodynamics likely to induce aneurysmal remodeling.

Landmark-Based Shape Analysis on Middle Cerebral Intracranial Aneurysms: A Geometric Morphometrics Approach to Infer Natural History.

BACKGROUND: Due to the scarcity of longitudinal data, the morphologic development of intracranial aneurysms (IAs) during their natural history remains poorly understood. However, longitudinal information can often be inferred from cross-sectional datasets as demonstrated by anatomists' use of geometric morphometrics to build evolutionary trees, reconstructing species inter-relationships based on morphologic landmarks. OBJECTIVE: We adopted these tools to analyze cross-sectional image data and infer relationships between IA morphologies. METHODS: On 3D reconstructions of 52 middle cerebral arteries (MCA) IAs (9 ruptured) and 10 IAfree MCAs (baseline geometries), 7 semi-automated landmarks were placed at the proximal parent artery and maximum height. From these, 64 additional landmarks were computationally generated to create a 71-landmark point cloud of 213 xyz coordinates. This data was normalized by Procrustes transformation and used in the principal component analysis, hierarchical clustering, and phylogenetic analyses. RESULTS: Principal component analysis showed separation of IA-free MCA geometries and grouping of ruptured IAs from unruptured IAs. Hierarchical clustering delineated a cluster of only unruptured IAs that were significantly smaller and more spherical than clusters that had ruptured IAs. Phylogenetic classification placed ruptured IAs more distally in the tree than unruptured IAs, indicating greater shape derivation. Groups of unruptured IAs were observed, but ruptured IAs were invariably found in mixed lineages with unruptured IAs, suggesting that some pathways of shape change may be benign while others are more associated with rupture. CONCLUSION: Geometric morphometric analyses of larger datasets may indicate particular pathways of shape change leading toward aneurysm rupture versus stabilization.

Whole blood transcriptome biomarkers of unruptured intracranial aneurysm.

BACKGROUND: The rupture of an intracranial aneurysm (IA) causes devastating subarachnoid hemorrhages, yet most IAs remain undiscovered until they rupture. Recently, we found an IA RNA expression signature of circulating neutrophils, and used transcriptome data to build predictive models for unruptured IAs. In this study, we evaluate the feasibility of using whole blood transcriptomes to predict the presence of unruptured IAs. METHODS: We subjected RNA from peripheral whole blood of 67 patients (34 with unruptured IA, 33 without IA) to next-generation RNA sequencing. Model genes were identified using the least absolute shrinkage and selection operator (LASSO) in a random training cohort (n = 47). These genes were used to train a Gaussian Support Vector Machine (gSVM) model to distinguish patients with IA. The model was applied to an independent testing cohort (n = 20) to evaluate performance by receiver operating characteristic (ROC) curve. Gene ontology and pathway analyses investigated the underlying biology of the model genes. RESULTS: We identified 18 genes that could distinguish IA patients in a training cohort with 85% accuracy. This SVM model also had 85% accuracy in the testing cohort, with an area under the ROC curve of 0.91. Bioinformatics reflected activation and recruitment of leukocytes, activation of macrophages, and inflammatory response, suggesting that the biomarker captures important processes in IA pathogenesis. CONCLUSIONS: Circulating whole blood transcriptomes can detect the presence of unruptured IAs. Pending additional testing in larger cohorts, this could serve as a foundation to develop a simple blood-based test to facilitate screening and early detection of IAs.

The asymmetric vascular stent: efficacy in a rabbit aneurysm model.

BACKGROUND AND PURPOSE: Development of hemodynamic modifying devices to treat intracranial aneurysms is an active area of research. The asymmetrical vascular stent (AVS), a stent containing a low-porosity patch, is such device. We evaluate AVS efficacy in an in vivo intracranial aneurysm model. METHODS: We created 24 elastase rabbit model aneurysms: 13 treated with the AVS, 5 treated with standard coronary stents, and 6 untreated controls. Four weeks after treatment, aneurysms underwent follow-up angiography, cone-beam micro-CT, histological evaluation, and selective electron microscopy scanning. RESULTS: Four rabbits died early in the study: 3 during AVS treatment and 1 control (secondary to intraprocedural vessel injury and an unrelated tumor, respectively). AVS-treated aneurysms exhibited very weak or no aneurysm flow immediately after treatment and no flow in all aneurysms at follow-up. Standard stent-treated aneurysms showed flow both after treatment (5/5) and at follow-up (3/5). All control aneurysms remained patent during the study. Micro-CT scans showed: 9 of 9 scanned AVS aneurysms were occluded, 6 of 9 AVS were ideally placed, and 3 of 9 low-porosity region partially covered the aneurysm neck; standard stent-treated aneurysms were 1 of 5 occluded, 2 of 5 patent, and 2 of 5 partially patent. Histology results demonstrated: for AVS-treated aneurysms, advanced thrombus organization in the (9/9); for standard stent-treated aneurysms, (1/4) no thrombus, (2/4) partially thrombosed, and (1/4) fully thrombosed; for control aneurysms (4/4), no thrombus. CONCLUSIONS: The use of AVS shows promise as a viable new therapeutic in intracranial aneurysm treatment. These data encourage further investigation and provide substantial support to the AVS concept.

The role of myosin II motor activity in distributing myosin asymmetrically and coupling protrusive activity to cell translocation.

Nonmuscle myosin IIA and IIB distribute preferentially toward opposite ends of migrating endothelial cells. To understand the mechanism and function of this behavior, myosin II was examined in cells treated with the motor inhibitor, blebbistatin. Blebbistatin at > or = 30 microM inhibited anterior redistribution of myosin IIA, with 100 microM blebbistatin causing posterior accumulation. Posterior accumulation of myosin IIB was unaffected. Time-lapse cinemicrography showed myosin IIA entering lamellipodia shortly after their formation, but failing to move into lamellipodia in blebbistatin. Thus, myosin II requires motor activity to move forward onto F-actin in protrusions. However, this movement is inhibited by myosin filament assembly, because whole myosin was delayed relative to a tailless fragment. Inhibiting myosin's forward movement reduced coupling between protrusive activity and translocation of the cell body: In untreated cells, body movement followed advancing lamellipodia, whereas blebbistatin-treated cells extended protrusions without displacement of the body or with a longer delay before movement. Anterior cytoplasm of blebbistatin-treated cells contained disorganized bundles of parallel microfilaments, but anterior F-actin bundles in untreated cells were mostly oriented perpendicular to movement. Myosin II may ordinarily move anteriorly on actin filaments and pull crossed filaments into antiparallel bundles, with the resulting realignment pulling the cell body forward.

Epigenetic landscapes suggest that genetic risk for intracranial aneurysm operates on the endothelium.

BACKGROUND: Genetics play an important role in intracranial aneurysm (IA) pathophysiology. Genome-wide association studies have identified several single nucleotide polymorphisms (SNPs) that are linked to IA but how they affect disease pathobiology remains poorly understood. We used Encyclopedia of DNA Elements (ENCODE) data to investigate the epigenetic landscapes surrounding genetic risk loci to determine if IA-associated SNPs affect functional elements that regulate gene expression and if those SNPs are most likely to impact a specific type of cells. METHODS: We mapped 16 highly significant IA-associated SNPs to linkage disequilibrium (LD) blocks within the human genome. Within these regions, we examined the presence of H3K4me1 and H3K27ac histone marks and CCCTC-binding factor (CTCF) and transcription-factor binding sites using chromatin immunoprecipitation-sequencing (ChIP-Seq) data. This analysis was conducted in several cell types relevant to endothelial (human umbilical vein endothelial cells [HUVECs]) and inflammatory (monocytes, neutrophils, and peripheral blood mononuclear cells [PBMCs]) biology. Gene ontology analysis was performed on genes within extended IA-risk regions to understand which biological processes could be affected by IA-risk SNPs. We also evaluated recently published data that showed differential methylation and differential ribonucleic acid (RNA) expression in IA to investigate the correlation between differentially regulated elements and the IA-risk LD blocks. RESULTS: The IA-associated LD blocks were statistically significantly enriched for H3K4me1 and/or H3K27ac marks (markers of enhancer function) in endothelial cells but not in immune cells. The IA-associated LD blocks also contained more binding sites for CTCF in endothelial cells than monocytes, although not statistically significant. Differentially methylated regions of DNA identified in IA tissue were also present in several IA-risk LD blocks, suggesting SNPs could affect this epigenetic machinery. Gene ontology analysis supports that genes affected by IA-risk SNPs are associated with extracellular matrix reorganization and endopeptidase activity. CONCLUSION: These findings suggest that known genetic alterations linked to IA risk act on endothelial cell function. These alterations do not correlate with IA-associated gene expression signatures of circulating blood cells, which suggests that such signatures are a secondary response reflecting the presence of IA rather than indicating risk for IA.

Nascent aneurysm formation at the basilar terminus induced by hemodynamics.

BACKGROUND AND PURPOSE: Hemodynamic insults at arterial bifurcations are hypothesized to play a key role in intracranial aneurysm formation. This study investigates aneurysm-initiating vascular responses at the rabbit basilar terminus subsequent to common carotid artery ligation. METHODS: Nine adult female New Zealand white rabbits were subjected to sham, unilateral, or bilateral common carotid artery ligation to produce varying degrees of compensatory basilar artery flow increase. Basilar artery flow velocity and geometry were monitored by transcranial Doppler and rotational angiography, respectively, for 12 weeks after surgery. Bifurcation tissues were harvested at 12 weeks and examined histologically. From the histological sections, we quantified the destructive structural changes at the basilar terminus and correlated them with the basilar artery flow rate increase. RESULTS: Subsequent to common carotid artery ligation, basilar artery flow rate increased by 105% to 900% at the maximum. All common carotid artery-ligated rabbits presented nascent aneurysm formation characterized by a bulge with thinned media and absent internal elastic lamina near the basilar terminus. We defined a nascent aneurysm index based on a multiplicative combination of the local destructive remodeling lengths measured at the nascent aneurysm. The nascent aneurysm index strongly correlated with the increase in basilar artery flow rate with R(2)=0.91. CONCLUSIONS: Without other known predisposition, flow increase alone at the basilar bifurcation can lead to a nascent aneurysm. This nascent aneurysm formation is dose-dependent on basilar artery flow increase.